Claims:

1. A method of detecting the presence of xenotropic MLV related virus
(XMRV) in an individual comprising:a) contacting a sample of the
individual with at least one set of primers wherein the set of primers
comprises:at least one forward primer and at least one reverse primer
which are complementary to all or a portion of an XMRV G1 gag nucleotide
sequence,at least one forward primer and at least one reverse primer
which are complementary to all or a portion of an XMRV G2 gag nucleotide
sequence,at least one forward primer and at least one reverse primer
which are complementary to all or a portion of an XMRV G3 gag nucleotide
sequence,at least one forward primer and at least one reverse primer
which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence,at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence,at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence,at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pol
nucleotide sequence,or a combination thereof;b) maintaining the sample
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences;c) detecting whether amplified
XMRV sequences are present in the sample;wherein if amplified XMRV
sequences are detected in the sample, then XMRV is present in the
individual.

3. The method of claim 1 wherein a polymerase chain reaction (PCR) is used
to amplify the primers.

4. The method of claim 3 wherein the PCR is real time PCR.

5. The method of claim 4 further comprising contacting the sample with one
or more fluorescently labeled probes.

6. The method of claim 5 wherein the probe is labeled with fluorescein.

7. The method of claim 5 wherein the forward primer complementary to all
or a portion of an XMRV G1 gag nucleotide sequence has a nucleotide
sequence comprising GGACTTTTTGGAGTGGCTTTGTT (SEQ ID NO: 1), the reverse
primer complementary to all or a portion of an XMRV G1 gag nucleotide
sequence has a nucleotide sequence comprising GCGTAAAACCGAAAGCAAAAT (SEQ
ID NO: 2) and the probe has a nucleotide sequence comprising
ACAGAGACACTTCCCGCCCCCG (SEQ ID NO: 3).

8. The method of claim 5 wherein the forward primer complementary to all
or a portion of an XMRV G2 gag nucleotide sequence has a nucleotide
sequence comprising GTAACTACCCCTCTGAGTCTAACCT (SEQ ID NO: 4), the reverse
primer complementary to all or a portion of an XMRV G3 gag nucleotide
sequence has a nucleotide sequence comprising CTTCTTGACATCCACAGACTGGTT
(SEQ ID NO: 5) and the probe has a nucleotide sequence comprising
TCCAGCGCATTGCATC (SEQ ID NO: 6).

9. The method of claim 5 wherein the forward primer complementary to all
or a portion of an XMRV G3 gag nucleotide sequence has a nucleotide
sequence comprising CTCAGGTCAAGTCTAGAGTGTTTTGT (SEQ ID NO: 7), the
reverse primer complementary to all or a portion of an XMRV G2 gag
nucleotide sequence has a nucleotide sequence comprising
CCTCCCAGGTGACGATATATGG (SEQ ID NO: 8) and the probe has a nucleotide
sequence comprising CCCCACGGACACCC (SEQ ID NO: 9).

10. The method of claim 5 wherein the forward primer complementary to all
or a portion of an XMRV P1 pol nucleotide sequence has a nucleotide
sequence comprising CGGGACAGAACTATCCAGTATGTGA (SEQ ID NO: 10), the
reverse primer complementary to all or a portion of an XMRV P1 pol
nucleotide sequence has a nucleotide sequence comprising
TGGCTTTGCTGGCATTTACTTG (SEQ ID NO: 11) and the probe has a nucleotide
sequence comprising ACCTGCACCGCCTGTG (SEQ ID NO: 12).

11. The method of claim 5 wherein the forward primer complementary to all
or a portion of an XMRV E1 env nucleotide sequence has a nucleotide
sequence comprising GGCCGAGAGAGGGCTACT (SEQ ID NO: 13), the reverse
primer complementary to all or a portion of an XMRV E1 env nucleotide
sequence has a nucleotide sequence comprising TGATGATGATGGCTTCCAGTATGC
(SEQ ID NO: 14) and the probe has a nucleotide sequence comprising
CACATCCCCATTTGCC (SEQ ID NO: 15).

12. The method of claim 5 wherein the forward primer complementary to all
or a portion of an XMRV E2 env nucleotide sequence has a nucleotide
sequence comprising CCCTAGTGGCCACCAAACAA (SEQ ID NO: 16), the reverse
primer complementary to all or a portion of an XMRV E2 env nucleotide
sequence has a nucleotide sequence comprising AAGGCCCCAAGGTCTGTATGT (SEQ
ID NO: 17) and the probe has a nucleotide sequence comprising
TCGAGCAGCTCCAGGCAGCCA (SEQ ID NO: 18).

13. The method of claim 5 wherein the forward primer complementary to all
or a portion of an XMRV E3 env nucleotide sequence has a nucleotide
sequence comprising TCAGGACAAGGGTGGTTTGAG (SEQ ID NO: 19), the reverse
primer complementary to all or a portion of an XMRV E3 env nucleotide
sequence has a nucleotide sequence comprising GGCCCATAATGGTGGATATCA (SEQ
ID NO: 20) and the probe has a nucleotide sequence comprising
TTAACAGGTCCCCATGGTTCACGACCA (SEQ ID NO: 21).

14. The method of claim 3 wherein the PCR is nested, reverse transcription
PCR comprising a first round of PCR and a second round of PCR.

15. The method of claim 14 whereinin the first round of PCR, the forward
primer for the gag sequence has a nucleotide sequence comprising
GAGTTCGTATTCCCGGCCGCAGC (SEQ ID NO: 24), the reverse primer for the gag
sequence has a nucleotide sequence comprising
GGTAACCCAGCGCCTCTTCTTGACATCC (SEQ ID NO: 25), the forward primer for the
env sequence has a nucleotide sequence comprising
CCCATGATGATGATGGCTTCCAGTATGC (SEQ ID NO: 22) and the reverse primer for
the env sequence has a nucleotide sequence comprising
GCTAATGCTACCTCCCTCCTGG (SEQ ID NO: 23); andin the second round of PCR,
the forward primer for the gag sequence has a nucleotide sequence
comprising ATCAGTTAACCTACCCGAGTCGGAC (SEQ ID NO: 28), the reverse primer
for the gag sequence has a nucleotide sequence comprising
GGTTTCGGCGTAAAACCGAAAGC (SEQ ID NO: 29), the forward primer for the env
sequence has a nucleotide sequence comprising GGGGACGATGACAGACACTTTCC
(SEQ ID NO: 26) and the reverse primer for the env sequence has a
nucleotide sequence comprising CACATCCCCATTTGCCACAGTAG (SEQ ID NO: 27).

16. The method of claim 1 wherein the amplified XMRV sequences are
detected using gel electrophoresis.

17. The method of claim 1 further comprising cloning the amplified XMRV
sequences.

18. The method of claim 1 further comprising comparing the amplified XMRV
sequences to a control.

19. The method of claim 1 wherein the sample is selected from the group
consisting of: urine, prostate tissue, prostatic fluids, bladder cancer
tissue or a combination thereof.

20. The method of claim 19 wherein the prostatic fluid is an expressed
prostate secretion (EPS).

21. The method of claim 20 wherein the EPS is semen.

22. The method of claim 1 wherein the individual is a human.

23. A method of detecting prostate cancer at an early stage in an
individual comprising:a) contacting a sample of the individual with at
least one set of primers wherein the set of primers comprises:at least
one forward primer and at least one reverse primer which are
complementary to all or a portion of an XMRV G1 gag nucleotide
sequence,at least one forward primer and at least one reverse primer
which are complementary to all or a portion of an XMRV G2 gag nucleotide
sequence,at least one forward primer and at least one reverse primer
which are complementary to all or a portion of an XMRV G3 gag nucleotide
sequence,at least one forward primer and at least one reverse primer
which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence,at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence,at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence,at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pol
nucleotide sequence,or a combination thereof;b) maintaining the sample
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences;c) detecting whether amplified
XMRV sequences are present in the sample;wherein the detection of
amplified XMRV sequences in the sample indicates that the individual has
prostate cancer at an early stage.

24. A method of detecting an individual at risk for developing prostate
cancer comprising:a) contacting a sample of the individual with at least
one set of primers wherein the set of primers comprises:at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV G1 gag nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV G2 gag nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV G3 gag nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E1 envelope nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E2 envelope nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E3 envelope nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV P1 Pol nucleotide sequence,or a combination
thereof;b) maintaining the sample under conditions which amplify the
primers if XMRV is present in the sample to produce amplified XMRV
sequences;c) detecting whether amplified XMRV sequences are present in
the sample;wherein the detection of amplified XMRV sequences in the
sample indicates that the individual is at risk for developing prostate
cancer.

25. A method of detecting recurrence of prostate cancer in an individual
comprising:a) contacting a sample of the individual with at least one set
of primers wherein the set of primers comprises:at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G1 gag nucleotide sequence,at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G2 gag nucleotide sequence,at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G3 gag nucleotide sequence,at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV E1 envelope nucleotide sequence,at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV E2 envelope nucleotide sequence,at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV E3 envelope nucleotide sequence,at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV P1 Pol nucleotide sequence,or a combination
thereof;b) maintaining the sample under conditions which amplify the
primers if XMRV is present in the sample to produce amplified XMRV
sequences;c) detecting whether amplified XMRV sequences are present in
the sample;wherein the detection of amplified XMRV sequences in the
sample indicates the recurrence of prostate cancer in the individual.

26. A method of monitoring a treatment of an individual that has prostate
cancer comprising:a) contacting a sample of the individual with at least
one set of primers wherein the set of primers comprises:at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV G1 gag nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV G2 gag nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV G3 gag nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E1 envelope nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E2 envelope nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E3 envelope nucleotide sequence,at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV P1 Pol nucleotide sequence;or a combination
thereof;b) maintaining the sample under conditions which amplify the
primers if XMRV is present in the sample to produce amplified XMRV
sequences;c) detecting whether amplified XMRV sequences are present in
the sample.

Description:

RELATED APPLICATION(S)

[0001]This application claims the benefit of U.S. Provisional Application
No. 61/203,556, filed on Dec. 23, 2008. The entire teachings of the above
application(s) are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003]Prostate cancer is the leading cause of non-cutaneous malignancies
and the second leading cause of cancer-related deaths among American men.
A need exists for improved methods of detection, particularly early
detection, of prostate cancer.

[0005]In one aspect, the invention is directed to a method of detecting
the presence of xenotropic MLV related virus (XMRV) in an individual. The
method comprises contacting a sample of the individual with at least one
set of primers wherein the set of primers comprises at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G1 gag nucleotide sequence, at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G2 gag nucleotide sequence, at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G3 gag nucleotide sequence, at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV E1 envelope nucleotide sequence, at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E2 envelope nucleotide sequence, at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E3 envelope nucleotide sequence, at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV P1 Pol nucleotide sequence, or a combination
thereof. The sample is maintained under conditions which amplify the
primers if XMRV is present in the sample to produce amplified XMRV
sequences. Whether amplified XMRV sequences are present in the sample are
detected, wherein if amplified XMRV sequences are detected in the sample,
then XMRV is present in the individual.

[0006]In another aspect, the invention is directed to method of detecting
prostate cancer (e.g., at an early stage) in an individual. The method
comprises contacting a sample of the individual with at least one set of
primers wherein the set of primers comprises at least one forward primer
and at least one reverse primer which are complementary to all or a
portion of an XMRV G1 gag nucleotide sequence, at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G2 gag nucleotide sequence, at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV G3 gag nucleotide sequence, at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV E1 envelope nucleotide sequence, at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E2 envelope nucleotide sequence, at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV E3 envelope nucleotide sequence, at least one
forward primer and at least one reverse primer which are complementary to
all or a portion of an XMRV P1 Pol nucleotide sequence, or a combination
thereof. The sample is maintained under conditions which amplify the
primers if XMRV is present in the sample to produce amplified XMRV
sequences, and whether amplified XMRV sequences are present in the sample
are detected. The detection of amplified XMRV sequences in the sample
indicates that the individual has prostate cancer at an early stage.

[0007]The invention also provides a method of detecting an individual at
risk for developing prostate cancer. The method comprises contacting a
sample of the individual with at least one set of primers wherein the set
of primers comprises at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G1 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G2 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G3 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pol
nucleotide sequence, or a combination thereof. The sample is maintained
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences, and whether amplified XMRV
sequences are present in the sample are detected. The detection of
amplified XMRV sequences in the sample indicates that the individual is
at risk for developing prostate cancer.

[0008]The invention also provides a method of detecting recurrence of
prostate cancer in an individual. The method comprises contacting a
sample of the individual with at least one set of primers wherein the set
of primers comprises at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G1 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G2 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G3 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pot
nucleotide sequence, or a combination thereof. The sample is maintained
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences, and whether amplified XMRV
sequences are present in the sample are detected. The detection of
amplified XMRV sequences in the sample indicates the recurrence of
prostate cancer in the individual.

[0009]The invention also provides a method of monitoring a treatment of an
individual that has prostate cancer. The method comprises contacting a
sample of the individual with at least one set of primers wherein the set
of primers comprises at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G1 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G2 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G3 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pol
nucleotide sequence; or a combination thereof. The sample is maintained
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences, and whether amplified XMRV
sequences are present in the sample are detected. The detection of
amplified XMRV sequences in the sample indicates that the treatment is
likely not effective or is likely not yet effective.

[0011]FIGS. 2A and 2B are standard curves of envelope RNA using E3 (FIG.
2A) and G3 (FIG. 2B) which was diluted to different dilutions and
analyzed by qRT/PCR using Ag-Path kit.

[0012]FIGS. 3A and 3B show the results of XMRV RNA copy number in urine of
prostate cancer patient VP663 using E1 site in env and E2 site in env,
respectively. Results are shown of qRT-PCR assays performed six times (x)
and in comparison to a standard curve generated with a 1.85 kb XMRV env
RNA produced by in vitro transcription. Y axis shows the Ct values, x
axis shows the log of the copy number.

[0013]FIG. 4 shows the results of detection of XMRV RNA in EPS of prostate
cancer patient VP 657 and VP 635 using E1 site in env. Results of qRT-PCR
assays were preformed in duplicate and are shown in comparison to a
standard curve generated with a 1.85 kb XMRV env RNA produced by in vitro
transcription. Y axis shows the Ct values, x axis shows the log of the
copy number.

[0014]FIG. 5 shows the amplification plot of qRT/PCR identification of
XMRV RNA in prostatitis patient using the E1 primer-probe combination.
Duplicate samples were assayed which shows very high Ct value
corresponding to very low copy number.

[0021]FIG. 11 is a gel of singleplex nested RT-PCR of RNA isolated from 3
prostate cancer patient urine samples, reaction time were done in
triplicates. Oligos 6200R and 5922F were used for the first round
followed by 6159R and 5942F for the second round of amplification.

[0022]FIG. 12 is a graph showing the detection and determination of XMRV
DNA copy numbers in DNA isolated from tumor-bearing prostate tissues of
men with the RNASEL QQ genotype following prostatectomy.

[0024]Specifically, described herein is the development of polymerase
chain reaction (PCR) assays (e.g., real-time quantitative RT-PCR
(qRT-PCR) assays) for the detection of XMRV nucleic acid in a sample
(e.g., urine and other bodily fluids, such as prostate secretions, and
semen) obtained from an individual (e.g., patient). In particular
aspects, highly sensitive, specific and quantitative real-time (RT) PCR
assays for XMRV nucleic acid (e.g., DNA; RNA) and nested RT-PCR assays
for detection of XMRV nucleic acid are described. These assays are useful
for determination of viral loads in tissues and fluids from individuals
with and without cancer. Also described herein is the correlation of the
prevalence and load of XMRV in prostate cancer cases with disease
parameters. XMRV is a newly discovered infection of tumor-bearing
prostate that correlates with mutations in a prostate cancer
susceptibility gene (RNASEL). The occurrence of XMRV infections in
prostate cancer cases provides for pathogenesis of the disease, assessing
risk, and novel therapeutic options.

[0025]In one aspect, total RNA was extracted from prostate tissue samples
using Trizol reagent (Invitrogen, Carlsbad, Calif.) and from urine and
prostatic secretion using the Magmax® Viral RNA Isolation Kit (Ambion
Inc., Austin Tex.) and then stored at -80° C. until further
processing. Initial screening of samples to detect XMRV gene sequences
was performed using qRT-PCR (performed on an Applied Biosystems 7500 Real
Time PCR system). In one embodiment, these reactions are performed using
a one-step RT-PCR reaction (AgPath-ID® kit, Applied Biosystems). PCR
assays for seven different regions in XMRV RNA have been developed (FIG.
1) which involved the design of sets of Taqman-based primers/probe used
to detect three regions in XMRV RNA, including one region of gag (G1) and
two regions of env (E1 & E2).

[0026]The presence of XMRV in prostatic secretions and urine as shown
herein is significant because such data provides a prostatic secretion-
and urine-based XMRV detection assay that is non-invasive, rapid, and
easy to perform, avoiding the morbidity and difficulty of obtaining blood
or tissue specimens for sampling. Current screening for prostate cancer
by prostate-specific antigen (PSA) levels and digital rectal exam often
does not begin until age 50 and has significant limitations and
inaccuracies. In contrast to these tests, the assays for XMRV described
herein can be performed on much younger men, especially those with a
family history of prostate cancer. Because men with XMRV infections,
especially those that fail to clear the virus, are likely at increased
risk of prostate cancer, these studies provide a new diagnostic for
evaluating risk of prostate cancer initiation or progression.

[0027]Accordingly, in one aspect, the invention is directed to a method of
detecting the presence of xenotropic MLV related virus (XMRV) in an
individual.

[0029]As used herein an "individual" refers to any subject in need of
screening. In particular embodiments, the individual is a mammal, such as
a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit,
guinea pig, rat, mouse or other bovine, ovine, equine, canine feline,
rodent or murine species). In one embodiment, the individual is a human.
In another embodiment, the individual is a human under the age of 50
years, 40 years, 30 years or 20 years. In another embodiment, the
individual is a cancer patient (e.g., a prostate cancer patient; and HPC
patient). In another embodiment, the individual is in remission from
prostate cancer. In another embodiment, the individual has or has had a
(one or more) XMRV infection. In another embodiment, the individual's
genome comprises a wild type, a heterozygous or a homozygous mutation of
the RNase L gene. In yet another embodiment, the individual expresses a
mutated or variant form of RNase L (e.g., R462Q; QQ RNASEL).

[0030]Although the invention is performed herein using urine and/or
prostatic secretion samples so as to demonstrate that the method is
non-invasive, rapid and easy to perform, one of skill in the art will
appreciate that any suitable biological sample obtained from an
individual can be used in the methods of the invention. The sample can be
a biological fluid, a tissue sample (e.g., prostate, bladder, seminal
glands, testes, kidney, bone marrow, colon, ileum, jejunum, pancreas,
adrenal glands, liver, heart, lung, spleen, brain cortex, brain stem,
cerebellum, inguinal lymph node, axillar lymph node and mesenteric lymph
node), a tumor sample (e.g., a prostate tumor, a bladder tumor, other
tumors of the male and female genitourinary tracts) and combinations
thereof. A suitable sample can be obtained for example by cell or tissue
biopsy. A sample can also be obtained from other tissues, bodily fluids
and products, e.g., from a tissue smear, tissue scrape, and the like.
Thus, the sample can be a biopsy specimen (e.g, tumor, polyp, mass
(solid, cellular)), aspirate, and/or smear sample). The sample can be
from a tissue that has a tumor (e.g., cancerous growth) and/or tumor
cells, or is suspected of having a tumor and/or tumor cells. For example,
a tumor biopsy can be obtained in an open biopsy, a procedure in which an
entire (excisional biopsy) or partial (incisional biopsy) mass is removed
from a target area. Alternatively, a tumor sample can be obtained through
a percutaneous biopsy, a procedure performed with a needle-like
instrument through a small incision or puncture (with or without the aid
of a imaging device) to obtain individual cells or clusters of cells
(e.g., a fine needle aspiration (FNA)) or a core or fragment of tissues
(core biopsy).

[0031]In a particular embodiment, the sample is a biological fluid.
Examples of a biological fluid that can be used in the methods include
urine, prostatic fluids, blood and semen. As used herein, "prostatic
fluids" include expressed prostate secretions (EPS) such as semen.

[0032]As described herein, the sample is contacted with at least one set
of primers and maintained under conditions which amplify the primers if
XMRV is present in the sample to produce amplified XMRV nucleic acid
sequences, also referred to herein as "amplicons" or "XMRV amplicons".
The amplified XMRV nucleic acid sequences can be, for example, XMRV DNA
or XMRV RNA.

[0033]A "set of primers" comprises at least one forward primer and at
least one reverse primer, wherein the forward primer and the reverse
primer in the set are complementary to all or a portion of an XMRV
nucleotide sequence (e.g., XMRV G1, XMRV G2, XMRV G3, XMRV P1, XMRV E1,
XMRV E2, XMRV E3). Typically, the forward primer and the reverse primer
within a set of primers are complementary to all or a portion of the same
region or a similar region of the XMRV nucleotide sequence (e.g., the gag
region, the env region, the pol region). As used herein, the term
"primer" refers to an oligonucleotide, which is capable of acting as a
point for the initiation of synthesis of a primer extension product that
is complementary to a target nucleotide sequence that is to be amplified,
referred to as the target or template nucleic acid sequence. In this
instance, the target or template nucleic acid sequence is all or a
portion (e.g., the gag region, the env region, the pol region) of an XMRV
nucleic acid sequence. The primer may occur naturally, as in a purified
restriction digest, or be produced synthetically. The appropriate length
of a primer depends on the intended use of the primer, but typically
ranges from about 5 to about 100; from about 5 to about 75; from about 5
to about 50; from about 5 to about 10; from about 10 to about 35; from
about 18 to about 22 nucleotides. A primer need not reflect the exact
sequence of the target sequence but must be sufficiently complementary to
hybridize with the target sequence for primer elongation to occur, i.e.,
the primer is sufficiently complementary to the target nucleotide
sequence such that the primer will anneal to the template under
conditions that permit primer extension. Reverse transcription can be
performed with M-MLV RT (such as Superscript® 1, II or III
(Invitrogen)) or Tth DNA polymerase in RT buffer using Oligo dT, random
hexamer or XMRV gene specific primers. As used herein, the phrase
"conditions that permit primer extension" refers to those conditions,
e.g., salt concentration (metallic and non-metallic salts), pH,
temperature, and necessary cofactor concentration, among others, under
which a given polymerase enzyme catalyzes the extension of an annealed
primer. Conditions for the primer extension activity of a wide range of
polymerase enzymes are known in the art. As one example, conditions
permitting the extension of a nucleic acid primer by Taq polymerase
include the following (for any given enzyme, there can and often will be
more than one set of such conditions): reactions are conducted in a
buffer containing 50 mM KCl, 10 mM Tris (pH 8.3-8.6), 1.5-4 mM
MgCl2, 200 μM of dNTPs; reactions can be performed at about
68°-72° C.

[0034]It will be clear to persons skilled in the art that the size of the
primer and the stability of hybridization will be dependent to some
degree on the ratio of A-T to C-G base pairings, since more hydrogen
bonding is available in a C-G pairing. Also, the skilled person will
consider the degree of homology between the extension primer to other
parts of the amplified sequence and choose the degree of stringency
accordingly. Guidance for such routine experimentation can be found in
the literature, for example, Molecular Cloning: a laboratory manual by
Sambrook, J., Fritsch E. F. and Maniatis, T. (1989) which is incorporated
herein by reference.

[0035]In addition to the primer pairs, probes can be included with
reporter dye at the 5' end (e.g., fluorescein, 6-carboxy fluorescein
(FAM), 6-FAM, 5-FAM, TAMRA) and quencher dye at the 3' end (e.g., BHQ-1,
BHQ-2, TAMRA, MGB) which will bind to the XMRV DNA during PCR (e.g., U.S.
Pat. No. 7,374,833 which is incorporated herein by reference).

[0036]For detection purposes, the primer can comprises at least one tag or
label. As used herein, "tag" or "label" are used interchangeably to refer
to any moiety that is capable of being specifically detected (e.g., by a
partner moiety), either directly or indirectly, and therefore, can be
used to identify and/or isolate a polynucleotide sequence that comprises
the tag. Suitable tags for the present invention include, among others,
affinity tags (e.g., biotin, avidin, streptavidin), haptens, ligands,
peptides, nucleic acids, fluorophores, chromophores, and epitope tags
that are recognized by an antibody (e.g., digoxigenin (DIG),
hemagglutinin (HA), myc, Flag) (Andrus, A. "Chemical methods for 5'
non-isotopic labelling of PCR probes and primers" (1995) in PCR 2: A
Practical Approach, Oxford University Press, Oxford, pp. 39-54). Other
suitable tags include, but are not limited to, chromophores,
fluorophores, haptens, radionuclides (e.g., 32P, 33P,
35S), fluorescence quenchers, enzymes, enzyme substrates, affinity
tags (e.g., biotin, avidin, streptavidin, etc.), mass tags,
electrophoretic tags and epitope tags that are recognized by an antibody.
In certain embodiments, the label is present on the 5 carbon position of
a pyrimidine base or on the 3 carbon deaza position of a purine base.

[0037]The primers have a nucleotide sequence that is complementary to all
or a portion of an XMRV sequence. In particular embodiments, the primers
have a nucleotide sequence that is complementary to all or a portion of
an XMRV gag sequence, an XMRV pol, an XMRV env sequence or a combination
thereof.

[0038]In one embodiment, at least one forward primer and at least one
reverse primer are complementary to all or a portion of an XMRV G1 gag
nucleotide sequence. As used herein, an "XMRV G1 gag nucleotide sequence"
refers to a sequence that is from about nucleotide 445 to about
nucleotide 528 of an XMRV genomic sequence. The length of the probe which
binds between two primers in the G1 gag nucleotide sequence can vary
between about 12 to about 40 nucleotides complementary to all or a
portion of XMRV G1 gag nucleotide sequence. Minor groove binding
principle can also be applied when the probe size is as short as about 12
nucleotides. In a particular embodiment, the forward primer complementary
to all or a portion of an XMRV G1 gag nucleotide sequence has a
nucleotide sequence comprising GGACTTTTTGGAGTGGCTTTGTT (SEQ ID NO: 1),
the reverse primer complementary to all or a portion of an XMRV G1 gag
nucleotide sequence has a nucleotide sequence comprising
GCGTAAAACCGAAAGCAAAAT (SEQ ID NO: 2) and the probe has a nucleotide
sequence comprising ACAGAGACACTTCCCGCCCCCG (SEQ ID NO: 3).

[0039]In another embodiment, at least one forward primer and at least one
reverse primer are complementary to all or a portion of an XMRV G2 gag
nucleotide sequence. As used herein, an "XMRV G2 gag nucleotide sequence"
refers to a sequence that is from about nucleotide 625 to about
nucleotide 708 of an XMRV genomic sequence. The length of probe which
binds between two primers in the G2 gag nucleotide sequence can vary
between about 12 to about 40 nucleotides complementary to all or a
portion of XMRV G1 gag nucleotide sequence. Minor groove binding can also
be applied when the probe size is as short as about 12 nucleotides. In a
particular embodiment, the forward primer complementary to all or a
portion of an XMRV G2 gag nucleotide sequence has a nucleotide sequence
comprising GTAACTACCCCTCTGAGTCTAACCT (SEQ ID NO: 4), the reverse primer
complementary to all or a portion of an XMRV G3 gag nucleotide sequence
has a nucleotide sequence comprising CTTCTTGACATCCACAGACTGGTT (SEQ ID NO:
5) and the probe has a nucleotide sequence comprising TCCAGCGCATTGCATC
(SEQ ID NO: 6).

[0040]In another embodiment, at least one forward primer and at least one
reverse primer are complementary to all or a portion of an XMRV G3 gag
nucleotide sequence. As used herein, an "XMRV G3 gag nucleotide sequence"
refers to a sequence that is from about nucleotide 797 to about
nucleotide 874 of an XMRV genomic sequence. The length of the probe which
binds between two primers in the G3 gag nucleotide sequence can vary
between about 12 to about 40 nucleotides complementary to all or a
portion of XMRV G1 gag nucleotide sequence. Minor groove binding
principle can also be applied when the probe size is as short as about 12
nucleotides. In a particular embodiment, the forward primer complementary
to all or a portion of an XMRV G3 gag nucleotide sequence has a
nucleotide sequence comprising CTCAGGTCAAGTCTAGAGTGTTTTGT (SEQ ID NO: 7),
the reverse primer complementary to all or a portion of an XMRV G2 gag
nucleotide sequence has a nucleotide sequence comprising
CCTCCCAGGTGACGATATATGG (SEQ ID NO: 8) and the probe has a nucleotide
sequence comprising CCCCACGGACACCC (SEQ ID NO: 9).

[0041]In another embodiment, at least one forward primer and at least one
reverse primer are complementary to all or a portion of an XMRV P1 Pol
nucleotide sequence. As used herein, an "XMRV P1 Pol nucleotide sequence"
refers to a sequence that is from about nucleotide 4843 to about
nucleotide 4912 of an XMRV genomic sequence. The length of the probe
which binds between two primers in the P1 pol nucleotide sequence can
vary between about 12 to about 40 nucleotides complementary to all or a
portion of XMRV G1 gag nucleotide sequence. Minor groove binding
principle can also be applied when the probe size is as short as about 12
nucleotides. In a particular embodiment, the forward primer complementary
to all or a portion of an XMRV P1 pol nucleotide sequence has a
nucleotide sequence comprising CGGGACAGAACTATCCAGTATGTGA (SEQ ID NO: 10),
the reverse primer complementary to all or a portion of an XMRV P1 pol
nucleotide sequence has a nucleotide sequence comprising
TGGCTTTGCTGGCATTTACTTG (SEQ ID NO: 11) and the probe has a nucleotide
sequence comprising ACCTGCACCGCCTGTG (SEQ ID NO: 12).

[0042]In another embodiment, at least one forward primer and at least one
reverse primer are complementary to all or a portion of an XMRV E1 env
nucleotide sequence. As used herein, an "XMRV E1 env nucleotide sequence"
refers to a sequence that is from about nucleotide 6142 to about
nucleotide 6197 of an XMRV genomic sequence. The length of the probe
which binds between two primers in the E1 env nucleotide sequence can
vary between about 12 to about 40 nucleotides complementary to all or a
portion of XMRV G1 gag nucleotide sequence. Minor groove binding
principle can also be applied when the probe size is as short as about 12
nucleotides. In a particular embodiment, the forward primer complementary
to all or a portion of an XMRV E1 env nucleotide sequence has a
nucleotide sequence comprising GGCCGAGAGAGGGCTACT (SEQ ID NO: 13), the
reverse primer complementary to all or a portion of an XMRV E1 env
nucleotide sequence has a nucleotide sequence comprising
TGATGATGATGGCTTCCAGTATGC (SEQ ID NO: 14) and the probe has a nucleotide
sequence comprising CACATCCCCATTTGCC (SEQ ID NO: 15).

[0043]In another embodiment, at least one forward primer and at least one
reverse primer are complementary to all or a portion of an XMRV E2 env
nucleotide sequence. As used herein, an "XMRV E2 env nucleotide sequence"
refers to a sequence that is from about nucleotide 7171 to about
nucleotide 7234 of an XMRV genomic sequence. The length of the probe
which binds between two primers in the E2 env nucleotide sequence can
vary between about 12 to about 40 nucleotides complementary to all or a
portion of XMRV G1 gag nucleotide sequence. Minor groove binding
principle can also be applied when the probe size is as short as about 12
nucleotides. In a particular embodiment, the forward primer complementary
to all or a portion of an XMRV E2 env nucleotide sequence has a
nucleotide sequence comprising CCCTAGTGGCCACCAAACAA (SEQ ID NO: 16), the
reverse primer complementary to all or a portion of an XMRV E2 env
nucleotide sequence has a nucleotide sequence comprising
AAGGCCCCAAGGTCTGTATGT (SEQ ID NO: 17) and the probe has a nucleotide
sequence comprising TCGAGCAGCTCCAGGCAGCCA (SEQ ID NO: 18).

[0044]In another embodiment, at least one forward primer and at least one
reverse primer are complementary to all or a portion of an XMRV E3 env
nucleotide sequence. As used herein, an "XMRV E3 env nucleotide sequence"
refers to a sequence that is from about nucleotide 7472 to about
nucleotide 7527 of an XMRV genomic sequence. The length of the probe
which binds between two primers in the E3 env nucleotide sequence can
vary between about 12 to about 40 nucleotides complementary to all or a
portion of XMRV G1 gag nucleotide sequence. Minor groove binding
principle can also be applied when the probe size is as short as about 12
nucleotides. In a particular embodiment, the forward primer complementary
to all or a portion of an XMRV E3 env nucleotide sequence has a
nucleotide sequence comprising TCAGGACAAGGGTGGTTTGAG (SEQ ID NO: 19), the
reverse primer complementary to all or a portion of an XMRV E3 env
nucleotide sequence has a nucleotide sequence comprising
GGCCCATAATGGTGGATATCA (SEQ ID NO: 20) and the probe has a nucleotide
sequence comprising TTAACAGGTCCCCATGGTTCACGACCA (SEQ ID NO: 21).

[0045]The primers are amplified using any suitable method known in the
art. As used herein, "amplification" or an "amplification reaction"
refers to any suitable method for amplification of a nucleic acid
sequence including polymerase chain reaction (PCR), ligase chain reaction
(LCR), rolling circle amplification (RCA), strand displacement
amplification (SDA) and multiple displacement amplification (MDA), as
will be understood by a person of skill in the art. Such methods for
amplification typically comprise, e.g., primers that anneal to the
nucleic acid sequence to be amplified, a DNA polymerase, and nucleotides.
Furthermore, amplification methods, such as PCR, can be solid-phase
amplification, polony amplification, colony amplification, emulsion PCR,
bead RCA, surface RCA, surface SDA, etc., as will be recognized by one of
skill in the art. It will also be recognized that it is advantageous to
use an amplification method that results in exponential amplification of
free DNA molecules in solution or tethered to a suitable matrix by only
one end of the DNA molecule. In addition, it will be recognized that it
is often advantageous to use amplification protocols that maximize the
fidelity of the amplified products to be used as templates in DNA
sequencing procedures. Such protocols use, for example, DNA polymerases
with strong discrimination against misincorporation of incorrect
nucleotides and/or strong 3' exonuclease activities (also referred to as
proofreading or editing activities) to remove misincorporated nucleotides
during polymerization.

[0046]In one embodiment, a PCR method is used to amplify the primers. As
known to those of skill in the art, PCR is a technique in which a DNA
polymerase is used to amplify a piece of DNA (e.g., a gene or portion
thereof; a non-coding region) by in vitro enzymatic replication. As PCR
progresses, the DNA generated is used as a template for replication which
sets in motion a reaction in which the DNA template is exponentially
amplified. With PCR a single or few copies of a piece of DNA are
amplified across several orders of magnitude, generating millions or more
copies of the DNA piece. As is also known in the art, PCR can be
extensively modified to perform a wide array of genetic manipulations.

[0047]PCR applications typically employ a heat-stable (thermostabile)
polymerase (e.g., DNA polymerase). A variety of polymerases for use in
PCR are known to this of skill in the art and include Taq polymerase, an
enzyme originally isolated from the bacterium Thermus aquaticus, and Vent
and Tth polymerases derived from microorganisms that normally reside at
high temperature. Consequently, these polymerase enzymes are quite stable
to heat denaturation, making them ideal enzymes for use in the polymerase
chain reaction. These polymerases, such as a DNA polymerase enzymatically
assembles a new DNA strand from DNA building blocks, the nucleotides, by
using single-stranded DNA as a template and DNA oligonucleotides (also
called DNA primers), which are required for initiation of DNA synthesis.

[0048]PCR methods typically use thermal cycling, i.e., alternately heating
and cooling the PCR sample to a defined series of temperature steps.
These thermal cycling steps physically separate the strands (at high
temperatures) in a e.g., DNA double helix (DNA melting) used as the
template during DNA synthesis (at lower temperatures) by the DNA
polymerase to selectively amplify the target DNA. Selectivity of PCR
arises from the use of primers that are complementary to the DNA region
targeted for amplification under specific thermal cycling conditions.

[0049]PCR typically involves the use of several components and reagents
such as a nucleic acid (e.g., DNA) template that contains the region
(target) to be amplified; one or more, typically two or more, primers
which are complementary to the nucleic acid regions at the 5' (five
prime) or 3' (three prime) ends of the nucleic acid region; one or more
polymerases e.g., with a temperature optimum at around 70° C.; one
or more deoxynucleoside triphosphates (dNTPs; also very commonly and
erroneously called deoxynucleotide triphosphates), the building blocks
from which the DNA polymerases synthesizes a new DNA strand; one or more
buffer solutions, providing a suitable chemical environment for optimum
activity and stability of the polymerase; one or more divalent cations,
e.g., magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can
be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+
concentration increases the error rate during DNA synthesis; and one or
more monovalent cation potassium ions.

[0050]PCR is commonly carried out in a reaction volume of 10-200 μl in
small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler which heats
and cools the reaction tubes to achieve the temperatures required at each
step of the reaction. Although one of skill in the art will appreciate
that PCR can occur in a variety of ways depending upon the desired
result(s), an example of a PCR can occur as follows. The PCR can begin
with an initialization step, which involves heating the reaction to a
temperature of about 94-96° C. (or about 98° C. if
extremely thermostable polymerases are used), which is held for about 1-9
minutes. This is typically used with DNA polymerases that require heat
activation by hot-start PCR. A denaturation step, which is the first
regular cycling event, involves heating the reaction to about
94-98° C. for about 20-30 seconds. This results in melting of DNA
template and primers by disrupting the hydrogen bonds between
complementary bases of the DNA strands, yielding single strands of DNA.
An annealing step, which involves lowering the temperature to about
50-65° C. for about 20-40 seconds allowing annealing of the
primers to the single-stranded DNA template, can then be carried out.
Typically the annealing temperature is about 3-5 degrees Celsius below
the melting temperature (Tm) of the primers used. Stable DNA-DNA hydrogen
bonds are generally formed when the primer sequence very closely matches
the template sequence. The polymerase binds to the primer-template hybrid
and begins DNA synthesis.

[0051]In the extension/elongation step, the temperature depends on the DNA
polymerase used; Taq polymerase has its optimum activity temperature at
about 75-80° C., and commonly a temperature of about 72° C.
is used with this enzyme. At this step the DNA polymerase synthesizes a
new DNA strand complementary to the DNA template strand by adding dNTPs
that are complementary to the template in 5' to 3' direction, condensing
the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end
of the nascent (extending) DNA strand. The extension time depends both on
the DNA polymerase used and on the length of the DNA fragment to be
amplified. A final elongation step is occasionally performed at a
temperature of about 70-74° C. for about 5-15 minutes after the
last PCR cycle to ensure that any remaining single-stranded DNA is fully
extended. A final hold step at about 4-15° C. for an indefinite
time can be employed for short-term storage of the reaction.

[0052]In a particular embodiment, a real time (RT/PCR) or quantitative,
real time PCR (qRT/PCR) reaction is used to amplify the primers if XMRV
is present in the sample. As understood by one of skill in the art,
RT/PCR DNA simultaneously quantifies and amplifies the nucleic acid. In
this method, the nucleic acid is specifically amplified by polymerase
chain reaction. After each round of amplification, the DNA is quantified.
Common methods of quantification include the use of fluorescent dyes that
intercalate with double-strand nucleic acid and modified oligonucleotides
(called probes) that fluoresce when hybridized with a complementary DNA.

[0053]Specifically, quantitative PCR (Q-PCR) is used to measure the
quantity of a PCR product (preferably real-time). The method
quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is
commonly used to determine whether a DNA sequence is present in a sample
and the number of its copies in the sample, and is also known as RT-PCR
(Real Time PCR), RQ-PCR, QRT-PCR or RTQ-PCR. RT-PCR commonly refers to
reverse transcription PCR, which can also be used in the methods
described herein, and is often used in conjunction with Q-PCR. QRT-PCR
methods use fluorescent dyes, such as Sybr Green, or
fluorophore-containing DNA probes, such as TaqMan, to measure the amount
of amplified product in real time.

[0054]Real-time polymerase chain reaction, also called quantitative real
time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain
reaction, is based on the polymerase chain reaction, which is used to
amplify and simultaneously quantify a targeted DNA molecule. It enables
both detection and quantification (as absolute number of copies or
relative amount when normalized to DNA input or additional normalizing
genes) of a specific sequence in a DNA sample.

[0055]The procedure follows the general principle of polymerase chain
reaction; its key feature is that the amplified DNA is quantified as it
accumulates in the reaction in real time after each amplification cycle.
Two common methods of quantification are the use of fluorescent dyes that
intercalate with double-stranded DNA, and modified DNA oligonucleotide
probes that fluoresce when hybridized with a complementary DNA.

[0056]Frequently, real-time polymerase chain reaction is combined with
reverse transcription polymerase chain reaction to quantify low abundance
messenger RNA (mRNA), enabling one of skill in the art to quantify
relative gene expression at a particular time, or in a particular cell or
tissue type. Although real-time quantitative polymerase chain reaction is
sometimes incorrectly abbreviated as RT-PCR, it should not be confused
with reverse transcription polymerase chain reaction, also known as
RT-PCR.

[0057]The reaction is typically run in a thermocycler as described herein,
and after each cycle, the levels of fluorescence are measured with a
detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR
product). With reference to a standard dilution, the dsDNA concentration
in the PCR can be determined.

[0058]A comparison of a measured DNA/RNA sample to a standard dilution
provides a fraction or ratio of the sample relative to the standard,
allowing relative comparisons between different tissues or experimental
conditions. The method can further comprise normalizing expression of a
target gene to a stably expressed gene.

[0059]In another embodiment, fluorescent reporter probes are used. A
sequence-specific RNA and/or DNA-based probe is used to quantify the
nucleic acid containing the probe sequence; therefore, use of the
reporter probe can increase specificity, and allow quantification even in
the presence of some non-specific DNA amplification. This allows for
multiplexing--assaying for several genes in the same reaction by using
specific probes with different-coloured labels, provided that all genes
are amplified with similar efficiency.

[0060]The reaction is typically carried out with an RNA-based probe with a
fluorescent reporter at one end and a quencher of fluorescence at the
opposite end of the probe. The close proximity of the reporter to the
quencher prevents detection of its fluorescence; breakdown of the probe
by the 5' to 3' exonuclease activity of the polymerase (e.g., taq
polymerase) breaks the reporter-quencher proximity and thus allows
unquenched emission of fluorescence, which can be detected. An increase
in the product targeted by the reporter probe at each PCR cycle therefore
causes a proportional increase in fluorescence due to the breakdown of
the probe and release of the reporter.

[0061]The PCR is prepared as usual, and the reporter probe is added. As
the reaction commences, during the annealing stage of the PCR both probe
and primers anneal to the DNA target. Polymerisation of a new DNA strand
is initiated from the primers, and once the polymerase reaches the probe,
its 5'-3-exonuclease degrades the probe, physically separating the
fluorescent reporter from the quencher, resulting in an increase in
fluorescence. Fluorescence is detected and measured in the real-time PCR
thermocycler, and its geometric increase corresponding to exponential
increase of the product is used to determine the threshold cycle (CT; Ct)
in each reaction.

[0062]In intact probes, reporter fluorescence is quenched. Probes and the
complementary DNA strand are hybridized and reporter fluorescence is
still quenched. During PCR, the probe is degraded by the polymerase and
the fluorescent reporter released.

[0063]Relative concentrations of DNA present during the exponential phase
of the reaction can be determined by plotting fluorescence against cycle
number on a logarithmic scale (so an exponentially increasing quantity
will give a straight line). A threshold for detection of fluorescence
above background is determined. The cycle at which the fluorescence from
a sample crosses the threshold is called the cycle threshold, Ct. Since
the quantity of DNA doubles every cycle during the exponential phase,
relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3
cycles earlier than another's has 23=8 times more template.

[0064]Amounts of RNA or DNA are then determined by comparing the results
to a standard curve produced by real-time PCR of serial dilutions (e.g.
undiluted, 1:4, 1:16, 1:64) of a known amount of RNA or DNA. As mentioned
above, to quantify gene expression, the measured amount of RNA from the
gene of interest is divided by the amount of RNA from a control sequence
(also referred to herein as a reference or housekeeping sequence) (e.g.,
gene) measured in the same sample to normalize for possible variation in
the amount and quality of RNA between different samples. This
normalization permits accurate comparison of expression of the sequence
of interest between different samples, provided that the expression of
the reference (housekeeping) sequence used in the normalization is very
similar across all the samples.

[0065]In another embodiment, nested, reverse transcription PCR is used to
amplify the primers. Nested PCR is a PCR with a second round of
amplification using a different set of primers. This second set of
primers is specific to a sequence found within the nucleotide sequence of
the initial conventional PCR amplicon. The use of a second amplification
step with the "nested" primer set results in a reduced background from
products amplified during the initial PCR due to the nested primers'
additional specificity to the region. The amount of amplicon produced is
increased as a result of the second round of amplification and due to a
reduction in any inhibitor concentrations. Reverse transcription, nested
PCR indicates that the reaction is initiated with DNA that has been
reverse transcribed from RNA.

[0066]As described herein, whether amplified XMRV sequences are present in
the sample are detected, wherein if amplified XMRV sequences are detected
in the sample, then XMRV is present in the individual. Detection of
amplified XMRV sequences can be achieved by resolving sequences by means
of, for example, gel electrophoresis (e.g., agarose gel), high-resolution
denaturing polyacrylamide/urea gel electrophoresis, capillary separation,
or other resolving means; followed by detecting the sequence using, for
example, a scanning spectrophotometer or fluorometer. In a particular
embodiment, fluorescently-labeled amplified XMRV sequences are resolved
by gel electrophoresis, according to procedures that are well known in
the art, and are subsequently detected in the gel using a standard
fluorometer. In one embodiment, a positive XMRV generates a band of 218
nucleotides in length, 112 nucleotides in length or a combination
thereof.

[0067]The method can further comprise determining the sequences of the
amplified XMRV sequence using procedures well known in the art.

[0068]As discussed above and as apparent to one of skill in the art, the
method of detecting the presence of XMRV in a sample can further comprise
the use of a control. That is, the amount or level of amplified XMRV
nucleic acid sequences in the sample can be compared to the amount or
level of amplified XMRV nucleic acid sequences in a control sample.
Suitable controls are well recognized in the art and include, for
example, a sample from an individual that is known to not be infected
with XMRV, a sample from an individual that is known to be infected with
XMRV, a sample from an individual that is a prostate cancer patient
(e.g., HPC patient), and/or a reference standard of authentic (positive)
XMRV RNA. The control sample can be the same type of sample as the sample
obtained from the individual (e.g., the sample obtained from the
individual and the control sample are urine samples) or the control
sample can be a different sample (e.g., the sample obtained from the
individual is a urine sample and the control sample is a tissue sample
such as a prostate tissue sample).

[0069]The methods for detecting XMRV is an individual can be used for a
variety of purposes such as for diagnostic and/or prognostic purposes for
predicting (or indicating) a clinical outcome (e.g., relapse, metastasis,
survival) of a newly diagnosed prostate cancer patient or a prostate
cancer patient that is undergoing or has undergone therapy.

[0070]Accordingly, the invention is directed to method of detecting
prostate cancer at an early stage in an individual. The method comprises
contacting a sample of the individual with at least one set of primers
wherein the set of primers comprises at least one forward primer and at
least one reverse primer which are complementary to all or a portion of
an XMRV G1 gag nucleotide sequence, at least one forward primer and at
least one reverse primer which are complementary to all or a portion of
an XMRV G2 gag nucleotide sequence, at least one forward primer and at
least one reverse primer which are complementary to all or a portion of
an XMRV G3 gag nucleotide sequence, at least one forward primer and at
least one reverse primer which are complementary to all or a portion of
an XMRV E1 envelope nucleotide sequence, at least one forward primer and
at least one reverse primer which are complementary to all or a portion
of an XMRV E2 envelope nucleotide sequence, at least one forward primer
and at least one reverse primer which are complementary to all or a
portion of an XMRV E3 envelope nucleotide sequence, at least one forward
primer and at least one reverse primer which are complementary to all or
a portion of an XMRV P1 Pol nucleotide sequence, or a combination
thereof. The sample is maintained under conditions which amplify the
primers if XMRV is present in the sample to produce amplified XMRV
sequences, and whether amplified XMRV sequences are present in the sample
are detected. The detection of amplified XMRV sequences in the sample
indicates that the individual has prostate cancer at an early stage.

[0071]The invention also provides a method of detecting an individual at
risk for developing prostate cancer. The method comprises contacting a
sample of the individual with at least one set of primers wherein the set
of primers comprises at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G1 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G2 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G3 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pot
nucleotide sequence, or a combination thereof. The sample is maintained
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences, and whether amplified XMRV
sequences are present in the sample are detected. The detection of
amplified XMRV sequences in the sample indicates that the individual is
at risk for developing prostate cancer.

[0072]The invention also provides a method of detecting recurrence of
prostate cancer in an individual. The method comprises contacting a
sample of the individual with at least one set of primers wherein the set
of primers comprises at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G1 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G2 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G3 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pol
nucleotide sequence, or a combination thereof. The sample is maintained
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences, and whether amplified XMRV
sequences are present in the sample are detected. The detection of
amplified XMRV sequences in the sample indicates the recurrence of
prostate cancer in the individual.

[0073]The invention also provides a method of monitoring a treatment of an
individual that has prostate cancer. The method comprises contacting a
sample of the individual with at least one set of primers wherein the set
of primers comprises at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G1 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G2 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV G3 gag
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E1 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E2 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV E3 envelope
nucleotide sequence, at least one forward primer and at least one reverse
primer which are complementary to all or a portion of an XMRV P1 Pol
nucleotide sequence; or a combination thereof. The sample is maintained
under conditions which amplify the primers if XMRV is present in the
sample to produce amplified XMRV sequences, and whether amplified XMRV
sequences are present in the sample are detected. The detection of
amplified XMRV sequences in the sample indicates that the treatment is
likely not effective or is likely not yet effective.

[0075]TaqMan® MGB (minor groove binder) primer probe combination is
obtained as a premix format (25× concentration) from Applied
Biosystems. For non-MOB primers and probes, the oligonucleotides were
resuspended to a stock concentration of 100 uM (100 picomoles/ul) in
1×Tris-EDTA (TE) buffer. Aliquots of 50 uM of primers and 10 uM of
probe working solution were made. The working probe was protected from
light by covering with aluminum foil.

[0085]Standard RNA and PCR precautions were used (e.g., used powder free
gloves, filter tips and clean area for RNA and PCR work).

Recommendations:

[0086]1. Freshly frozen prostate tissue was the best source for XMRV
identification. Paraffin embedded tissue was not a good source.

[0087]2. During the prostatectomy in the operating room, the prostate
juice was collected in RNase free labeled tubes and immediately frozen in
dry ice.

[0088]3. Glass homogenizer were not used to mince the frozen prostate
tissues to avoid cross contamination.

[0089]4. When possible, a separate space for dealing with prostate RNA was
designated.

[0090]5. Preferably separate pipettes were used to add reagents, which
were not used to pipette high copy standard plasmid or RNA.

[0091]6. Preferably, at least two replicates of qRT/PCR were performed on
various patient RNA samples.

RNA:

[0092]1. RNA was isolated from prostate tissue or from prostate secretion.

[0093]2. In vitro transcribed XMRV RNA (XMRV VP62 RNA sequence between
nucleotides 5761 and 7691) was used for detection of Env RNA or in vitro
transcribed XMRV RNA (XMRV VP62 RNA sequence between nucleotides 1 and
991 for detection of Gag RNA.

Protocol:

[0094]Isolation of RNA from Prostate Tissue

[0095]The standard RNA isolation method from prostate tissues using Trizol
reagent following manufacturer's instruction was used. The required
amount of Trizol was added to a clean Petri-dish and the frozen tissue
was minced directly in the reagent using disposable forceps. The yield
was about 15-20 ugs RNA from <1 cm3 prostate tissue.

Isolation of RNA in Expressed Prostate Secretion (EPS) and Urine

[0096]The prostatic fluids were collected in RNAse-free microfuge tubes by
manually milking secretions from the prostate after the prostate was
removed during surgery, flash frozen and stored at -80° C. until
RNA isolation. The RNA isolation from 100-200 μl samples was performed
using MagMAX® Viral RNA Isolation kit (Ambion, Tex., USA) with some
modifications as stated. For each isolation, the EPS sample was added to
602 μl of Lysis/Binding solution containing 300 μl of Lysis/Binding
solution concentrate, 2 μl of carrier RNA and 300 μl of
isopropanol. This was followed by addition of 40 μl of Bead Mix
containing 20 μl of RNA binding beads and 20 μl of Lysis/Binding
enhancer. The washing step was performed following manufacturer's
protocol and eluted in 40-60 μl of preheated elution buffer.
Generally, the amount of RNA obtained was in the range of 50-100 ng/μl
as assessed using NanoDrop® ND 1000 Spectrophotometer (NanoDrop
Technologies).

qRT/PCR using AgPath-ID® Kit

[0097]In one aspect, a two primer probe combination in separate reactions
was used for the detection of XMRV in RNA isolated from a patient sample.

[0098]1. The standard RNA and prostate RNA were thawed in two different
ice buckets to avoid cross contamination during addition.

[0099]2. At least six different dilution of RNA (10 fold each dilution) in
RNA storage solution was made.

[0100]3. All the reagents of AgPath-ID® kit were thawed on ice except
the enzyme).

[0103]While using MGB probe, 1.25 ul of the probes was used instead of
individual primers and probe.

Instrument Set Up:

[0104]Set up the instrument as recommended by the manufacturer.

[0105]Condition:

[0106]Stage 1, Rep=1

[0107]Step 1: 45° C. for 10 mins

[0108]Stage 2, Rep=1

[0109]Step 1: 95° C. for 10 mins

[0110]Stage 3, Rep=55 ***

[0111]Step 1: 95° C. for 0.15 min

[0112]Step 2: 58° C. for 1.0 min (Data Collection)

[0113]* * * Generally, the prostate RNA has very low copy of XMRV RNA with
Ct value of >35.

Data Analysis:

[0114]Save the data and transfer to lab computer. Transfer the data to
Excel spreadsheet for analysis. The graph with the standard RNA should
generate R2 value of >0.98 and slope ˜-3.3 to -3.5.

Results

[0115]Standard gag and Envelope RNA was diluted to different dilution and
performed qRT/PCR using Ag-Path kit. The standard curve results are shown
in FIGS. 2A and 2B.

[0116]FIGS. 3A and 3B shows the results of XMRV RNA copy number in urine
of prostate cancer patient VP663 using E1 and E2 sites in env,
respectively. Results of qRT-PCR assays were performed six times (x) and
is shown in comparison to a standard curve generated with a 1.85 kb XMRV
env RNA produced by in vitro transcription. Y axis shows the Ct values, x
axis shows the log of the copy number.

[0117]FIG. 4 shows qRT/PCR identification of XMRV RNA in prostate cancer
patients (VP 635 and VP 657) expressed prostate secretions (EPS) by
manual milking of the prostate during radical prostatectomy. Assays were
done in duplicate as shown in comparison to a standard curve generated
with a 1.85 kb XMRV env RNA produced by in vitro transcription. Y axis
shows the Ct values, x axis shows that log of the copy number.

[0118]FIG. 5 shows the amplification plot of qRT-PCR analysis of XMRV RNA
isolated from prostatitis patient's EPS (Patient P1). Very high Ct
corresponds to low copy of XMRV RNA in the sample. In no template control
sample, water was added in the reaction instead of RNA.

[0119]FIGS. 6A and 6B show the amplification of plot qRT-PCR analysis of
XMRV RNA isolated from prostate cancer patients' EPS (pj 339, pj 301, pj
302, pj 304). Only one dilution of three positive standard RNA was used
in the reaction.

Example 2

Detection of XMRV DNA by qPCR

Reagents

[0120]QIAamp DNA mini kit (Qiagen Cat: 51306)

TaqMan® Universal PCR Master Mix (Applied Biosystems Cat:430-4437)

Protocol:

[0121]Using sterile forceps and scalpel, a slice of frozen prostate tissue
was cut. The tissue slice was minced on a petri dish. DNA was isolated
using a standard protocol from the QIAamp DNA mini kit. The DNA was
alcohol precipitated, washed with 70% ethanol and resuspended in 20 μl
of TE. About 250-500 ngs of DNA was used in each reaction.

[0130]1. A clean area of the lab dedicated for patient samples only was
assigned. High copy XMRV nucleic acids should not be present in this
area.

[0131]2. During nested PCR, extreme precaution was taken to avoid
cross-contamination from positive samples. It is advisable to standardize
the PCR of positive control before the experimental sample. Use the
condition for patient sample without any positive control.

[0132]3. Preferably separate pipettes were used to add reagents, which
were not used to pipette high copy standard plasmid or RNA.

[0133]Standard RNA and PCR precautions were taken (e.g., powder free
gloves, filter tips and clean area for RNA and PCR work).

[0151]After the PCR, 8 ul of the product was loaded on to 2% Agarose gel
and the band was visualized under UV transilluminator. A positive RNA
generated bands of 218 and 112 nucleotides in length. In the upper panel
of FIG. 7, the location of the primers used for multiplex RT-PCR of XMRV
is shown. In the lower panel of FIG. 7, a gel of multiplex RT-PCR, which
was performed using 3000 to 30 copies of XMRV RNA along with RNA isolated
from a prostate cancer patient EPS (pj339), is shown. The respective
sequences are shown in FIG. 10.

[0152]In one aspect, the PCR products are gel purified and the sequence is
verified.

[0153]FIG. 8 is a gel of singleplex nested RT-PCR of RNA isolated from 6
prostate cancer patient urine samples using Tth and HotStart Polymerase
following the protocol described above. Oligos 6200R and 5922F were used
for the first round followed by 6159R and 5942F for the second round of
amplification.

[0154]FIG. 9 is a gel of singleplex nested RT-PCR of RNA isolated from 17
prostate cancer patient expressed secretions during prostatectomy. Oligos
6200R and 5922F were used for the first round followed by 6159R and 5942F
for the second round of amplification.

[0155]FIG. 11 is a gel of singleplex nested RT-PCR of RNA isolated from 3
prostate cancer patient urine samples, reaction time were done in
triplicates. Oligos 6200R and 5922F were used for the first round
followed by 6159R and 5942F for the second round of amplification.

SUMMARY

[0156]Prostate tissue, urine and prostatic secretions were collected from
patients. For patients with prostate cancer, urine samples were obtained
immediately prior to surgery. Prostatic fluid was also collected from the
same patients at the time of prostatectomy by manually milking secretions
from the prostate and seminal vesicles once the specimen had been removed
from the patient. Approximately 50 prostate secretions and a similar
number of urine samples from men with prostate cancer were assayed. About
20 bladder cancer tissue samples were also assayed and none were positive
for XMRV. Regions of two XMRV genes (gag and env) were assayed in
duplicate or triplicate. Evidence of XMRV in the prostatic tissue,
prostatic secretions and urine of several men with prostate cancer has
been demonstrated by simultaneously detecting XMRV gag and env sequences
through qRT-PCR. A subset of these samples were confirmed by sequencing
of the amplified regions of qRT/PCR respectively. In 5 cases, XMRV gag
sequences isolated from the patient's urine and prostatic fluid were
sequenced following PCR and found to be 100% identical to each other. The
gag fragment also shared 100% homology with that of two XMRV strains,
VP62 (GenBank Accession No. DQ399707) and VP35 (GenBank Accession No.
DQ241301), and shared 98% homology with XMRV VP42 (GenBank Accession No.
DQ241302). All three were from men with the QQ RNASEL genotype. In five
cases a 218 nt region of the XMRV env gene was also identified by nested
RT-PCR with a sequence that was identical with that of previously
published strains for XMRV. An example of the detection of XMRV RNA by
qRT-PCR in urine from a QQ RNASEL prostate cancer patient is presented
(FIG. 3A). An example of a nested XMRV env RT-PCR product from a
prostatic secretion isolated from a prostate cancer case with the RNASEL
QQ genotype is shown (FIG. 3A). Detection and determination of XMRV DNA
copy numbers were also determined in DNA isolated from tumor-bearing
prostate tissues of men with the RNASEL QQ genotype following
prostatectomy (Example 2, FIG. 12).

[0157]The teachings of all patents, published applications and references
cited herein are incorporated by reference in their entirety.

[0158]While this invention has been particularly shown and described with
references to example embodiments thereof, it will be understood by those
skilled in the art that various changes in form and details may be made
therein without departing from the scope of the invention encompassed by
the appended claims.